Metabolic engineering for higher alcohol production.

Engineering microbial hosts for the production of higher alcohols looks to combine the benefits of renewable biological production with the useful chemical properties of larger alcohols. In this review we outline the array of metabolic engineering strategies employed for the efficient diversion of carbon flux from native biosynthetic pathways to the overproduction of a target alcohol. Strategies for pathway design from amino acid biosynthesis through 2-keto acids, from isoprenoid biosynthesis through pyrophosphate intermediates, from fatty acid biosynthesis and degradation by tailoring chain length specificity, and the use and expansion of natural solvent production pathways will be covered.

Rolling circle amplification of synthetic DNA accelerates biocatalytic determination of enzyme activity relative to conventional methods.

The ability to quickly and easily assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis. Cell-free synthesis, although not practically amenable for large-scale enzyme production, provides a way to accelerate the timeline for screening enzyme candidates using small-scale reactions. However, because cell-free enzyme synthesis requires a considerable amount of template DNA, the preparation of high-quality DNA "parts" in large quantities represents a costly and rate-limiting prerequisite for high throughput screening. Based on time-cost analysis and comparative activity data, a cell-free workflow using synthetic DNA minicircles and rolling circle amplification enables comparable biocatalytic activity to cell-based workflows in almost half the time. We demonstrate this capability using a panel of sequences from the carbon-nitrogen hydrolase superfamily that represent possible green catalysts for synthesizing small molecules with less waste compared to traditional industrial chemistry. This method provides a new alternative to more cumbersome plasmid- or PCR-based protein expression workflows and should be amenable to automation for accelerating enzyme screening in industrial applications.

Systematic Approaches to Efficiently Produce 2,3-Butanediol in a Marine Cyanobacterium.

Cyanobacteria have attracted significant interest as a platform for renewable production of fuel and feedstock chemicals from abundant atmospheric carbon dioxide by way of photosynthesis. While great strides have been made in developing this technology in freshwater cyanobacteria, logistical issues remain in scale-up. Use of the cyanobacterium Synechococcus sp. PCC 7002 (7002) as a chemical production chassis could address a number of these issues given the higher tolerance to salt, light, and heat as well as the fast growth rate of 7002 in comparison to traditional model cyanobacteria such as Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803. However, despite growing interest, the development of genetic engineering tools for 7002 continues to lag behind those available for model cyanobacterial strains. In this work we demonstrate the systematic development of a 7002 production strain for the feedstock chemical 2,3-butanediol (23BD). We expand the range of tools available for use in 7002 by identifying and utilizing new integration sites for homologous recombination, demonstrating the inducibility of theophylline riboswitches, and screening a set of isopropyl ß-d-1-thiogalactopyranoside (IPTG) inducible promoters. We then demonstrate improvements of 23BD production with the systematic screening of different conditions including: operon arrangement and copy number, light strength, inducer concentration, cell density at the time of induction, and nutrient concentration. Final production tests yielded titers of 1.6 g/L 23BD after 16 days at a rate of 100 mg/L/day. This work represents great strides in the development of 7002 as an industrially relevant production host.

Cyanobacterial metabolic engineering for biofuel and chemical production.

Rising levels of atmospheric CO2 are contributing to the global greenhouse effect. Large scale use of atmospheric CO2 may be a sustainable and renewable means of chemical and liquid fuel production to mitigate global climate change. Photosynthetic organisms are an ideal platform for efficient, natural CO2 conversion to a broad range of chemicals. Cyanobacteria are especially attractive for these purposes, due to their genetic malleability and relatively fast growth rate. Recent years have yielded a range of work in the metabolic engineering of cyanobacteria and have led to greater knowledge of the host metabolism. Understanding of endogenous and heterologous carbon regulation mechanisms leads to the expansion of productive capacity and chemical variety. This review discusses the recent progress in metabolic engineering of cyanobacteria for biofuel and bulk chemical production since 2014.

Genome Engineering of the 2,3-Butanediol Biosynthetic Pathway for Tight Regulation in Cyanobacteria.

Cyanobacteria have gained popularity among the metabolic engineering community as a tractable photosynthetic host for renewable chemical production. However, though a number of successfully engineered production systems have been reported, long-term genetic stability remains an issue for cyanobacterial systems. The genetic engineering toolbox for cyanobacteria is largely lacking inducible systems for expression control. The characterization of tight regulation systems for use in cyanobacteria may help to alleviate this problem. In this work we explore the function of the IPTG inducible promoter P(L)lacO1 in the model cyanobacterium Synechococcus elongatus PCC 7942 as well as the effect of gene order within an operon on pathway expression. According to our experiments, P(L)lacO1 functions well as an inducible promoter in S. elongatus. Additionally, we found that gene order within an operon can strongly influence control of expression of each gene.

Cyanobacteria as a Platform for Biofuel Production.

Cyanobacteria have great potential as a platform for biofuel production because of their fast growth, ability to fix carbon dioxide gas, and their genetic tractability. Furthermore they do not require fermentable sugars or arable land for growth and so competition with cropland would be greatly reduced. In this perspective we discuss the challenges and areas for improvement most pertinent for advancing cyanobacterial fuel production, including: improving genetic parts, carbon fixation, metabolic flux, nutrient requirements on a large scale, and photosynthetic efficiency using natural light.